The Stress-Induced Soybean NAC Transcription Factor GmNAC81 Plays a Positive Role in Developmentally Programmed Leaf Senescence Maiana Reis Pimenta 1,3 , Priscila Alves Silva 1 , Giselle Camargo Mendes 1 , Janaı ´na Roberta Alves 1,2 , Hanna Durso Neves Caetano 1,2 , Joao Paulo Batista Machado 1,2 , Otavio Jose ´ Bernardes Brustolini 1,2 , Paola Avelar Carpinetti 1,2 , Bruno Paes Melo 1,2 , Jose ´ Cleydson Ferreira Silva 1,2 , Gustavo Lea ˜o Rosado 1,2 , Ma ´rcia Flores Silva Ferreira 1,4 , Maximillir Dal-Bianco 1,2 , Edgard Augusto de Toledo Picoli 3 , Francisco Jose ´ Lima Aragao 5 , Humberto Josue ´ Oliveira Ramos 1,2 and Elizabeth Pacheco Batista Fontes 1,2,* 1 National Institute of Science and Technology in Plant-Pest Interactions, Universidade Federal de Vic ¸osa, 36570.000, Vic ¸osa, MG, Brazil 2 Departamento de Bioquı ´mica e Biologia Molecular/Bioagro, Universidade Federal de Vic ¸osa, 36570.000, Vic ¸osa, MG, Brazil 3 Departamento de Biologia Vegetal, Universidade Federal de Vic ¸osa, 36570.000, Vic ¸osa, MG, Brazil 4 Departamento de Biologia, Universidade Federal do Espı ´rito Santo, 29500.000, Alegre, ES, Brazil 5 Embrapa Recursos Gene ´ticos e Biotecnologia, PqEB W5 Norte, 70770-900, Brası ´lia, DF, Brazil *Corresponding author: E-mail, bbfontes@ufv.br; Fax, +55-31-3899-2864. (Received April 13, 2015; Accepted March 15, 2016) The onset of leaf senescence is a highly regulated develop- mental change that is controlled by both genetics and the environment. Senescence is triggered by massive transcrip- tional reprogramming, but functional information about its underlying regulatory mechanisms is limited. In the current investigation, we performed a functional analysis of the soybean (Glycine max) osmotic stress- and endoplasmic reticulum (ER) stress-induced NAC transcription factor GmNAC81 during natural leaf senescence using overexpres- sion studies and reverse genetics. GmNAC81-overexpressing lines displayed accelerated flowering and leaf senescence but otherwise developed normally. The precocious leaf senescence of GmNAC81-overexpressing lines was asso- ciated with greater Chl loss, faster photosynthetic decay and higher expression of hydrolytic enzyme-encoding GmNAC81 target genes, including the vacuolar processing enzyme (VPE), an executioner of vacuole-triggered pro- grammed cell death (PCD). Conversely, virus-induced gene silencing-mediated silencing of GmNAC81 delayed leaf senescence and was associated with reductions in Chl loss, lipid peroxidation and the expression of GmNAC81 direct targets. Promoter–reporter studies revealed that the expression pattern of GmNAC81 was associated with senescence in soybean leaves. Our data indicate that GmNAC81 is a positive regulator of age-dependent senes- cence and may integrate osmotic stress- and ER stress- induced PCD responses with natural leaf senescence through the GmNAC81/VPE regulatory circuit. Keywords: Developmental regulation  GmNAC81  Glycine max  Leaf senescence  NAC family  Transcription factor. Abbreviations: DAG, days after germination; ER, endoplas- mic reticulum; GmERD15, Glycine max early responsive to dehydration 15; GUS, b-glucuronidase; JA, jasmonic acid; MDA, malondialdehyde; MeJA, methyl jasmonate; NAC, no apical meristem (NAM), Arabidopsis ATAF1/2, and cup- shaped cotyledon (CUC); NRP/DCD, developmental cell death (DCD) domain-containing N-rich protein (NRP); PCD, programmed cell death; qRT–PCR, quantitative reverse transcription–PCR; SA, salicylic acid; SAG, senescence- associated gene; SoCSV, Soybean chlorotic spot virus; TBA, thiobarbituric acid; TF, transcription factor; VIGS, virus- induced gene silencing; VPE, vacuolar processing enzyme. Introduction Leaf senescence is a developmental process that begins with the transition of a photosynthetic leaf organ to an actively degen- erating, nutrient-recycling leaf tissue. Senescence ensures the recycling of resources from senescent leaves to other plant organs to support growth and reproduction, and it becomes visible via leaf yellowing due to Chl loss. Leaf senescence is genetically linked to aging and involves the regulated expres- sion of senescence-associated genes (SAGs; Yoshida 2003, Lim et al. 2007). Several SAGs also respond to environmental stres- sors and may integrate stress-induced regulatory cascades with developmental signals (Balazadeh et al. 2010). The massive changes in gene expression that accompany the onset of leaf senescence are probably controlled by a subset of senescence- associated transcription factors (TFs), but their potential func- tions and the regulatory cascades that they control are largely unknown. Recently, high-resolution temporal expression pro- files revealed that a large fraction of NAC [for no apical meri- stem (NAM), Arabidopsis ATAF1/2 and cup-shaped cotyledon (CUC)] TFs are differentially expressed during several stages of natural leaf senescence in Arabidopsis (Breeze et al. 2011), sug- gesting that they play a crucial role in the regulation of senes- cence; however, functional information is available for only a few family members (Guo and Gan 2006, Kim et al. 2009, Plant Cell Physiol. 57(5): 1098–1114 (2016) doi:10.1093/pcp/pcw059, Advance Access publication on 25 March 2016, available online at www.pcp.oxfordjournals.org ! The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com Regular Paper Downloaded from https://academic.oup.com/pcp/article-abstract/57/5/1098/2223406 by Universidade Federal de Viçosa user on 16 April 2018